Marburg I mutant of factor VII activating protease (FSAP) as risk factor for arterial thrombosis

ABSTRACT

A novel arterial thrombosis risk factor comprising one or more of the identified mutants of coagulation factor VII-activating protease (FSAP) is described. In addition, diagnostic determination methods for detecting these mutants which are identified as risk factors are described.

[0001] This application is a continuation-in-part of application Ser.No. 09/912,559, which claims priority to German Application Nos. 100 36641.4, filed Jul. 26, 2000, 100 50 040.4, filed Oct. 10, 2000, 100 52319.6 filed Oct. 21, 2000, and 101 18 706.8, filed Apr. 12, 2001. Thisapplication also claims priority to German Application Nos. DE 102 12246.6, filed Mar. 19, 2002; and DE DE 102 38 429.0, filed Aug. 16, 2002.All of the above-listed applications, in their entirety, areincorporated herein by reference.

[0002] The invention relates to mutants of factor VII-activatingprotease (FSAP) and to reduced blood plasma levels of FSAP as indicatorsof an increased risk for the development and progression ofatherothrombosis (or arterial thrombosis) and of the pathophysiologicalsequelae resulting therefrom.

[0003] Atherosclerosis is a pathological change in the arteries which isassociated inter alia with hardening, thickening and loss of elasticitythereof and is regarded as the main cause of myocardial infarction andstroke and of other disorders. Numerous exogenous and endogenous factorsare thought to be responsible for the initiation and progression ofatherosclerosis, for example hypertension, hyperlipidemia, diabetes,toxins, nicotine, excessive alcohol consumption and inflammations. Theseinfluences are referred to as risk factors. However, with the increasingnumber of studies and improved analytical methods, in recent yearsfurther risk factors for atherosclerosis and subsequent arterialthrombosis have been found.

[0004] Risk factors are investigated in epidemiological studies, as inthe Bruneck study which has become well known among specialists in thefield. One thousand inhabitants of Bruneck, Italy, were recruited forthis study in 1990. Ultrasound investigations of the carotid artery andanalyses of a number of blood parameters and questioning of the subjectsmade it possible to establish a broad database for further follow-up ofthe development and progression of atherosclerosis. These investigationswere continued on the same subjects and analyzed at 5-year intervals. Amodel of the development of atherosclerosis and its progression wasderived therefrom. As a first result of this study, a connection wasfound between the development of atherosclerosis and known, traditionalrisk factors such as the aforementioned hyperlipidemia and otherfactors. However, if the atherosclerotic plaque reaches such an extentthat the blood vessel is occluded by up to 40%, other risk factorsbecome important and may significantly influence the further progress ofthe atherosclerosis and the vascular occlusion. These factors include inparticular plasma proteins which intervene in hemostasis. A reducedcoagulation-inhibitory potential contributes to this, e.g. reducedantithrombin or protein C levels, or the so-called APC (activatedprotein C) resistance. A reduction in the fibrinolytic potential maytherefore have a crucial influence on the progression of the vascularocclusion, as is observed for example when the levels of lipoprotein (a)in the blood are raised.

[0005] To date, risk factors for venous occlusive disorders have beenidentified, such as APC resistance (Factor V Leiden). The extent towhich a given risk factor increases the risk for a condition may beexpressed as an “odds ratio.” The odds ratio for venous occlusivedisorders in heterozygotes for the APC resistance mutation has beenreported to be about 5 to 8, compared with subjects without APCresistance. For arterial occlusive disorders, on the other hand, thisrisk factor has been reported with low odds ratios of average about 2.

[0006] The plasma samples and DNAs available from the subjects in theBruneck study were therefore investigated once again for the presence ofother risk factors for atherosclerosis, directing particular attentionat recently found mutants of coagulation factor VII-activating protease(=FSAP), referred to hereinafter as the FSAP Marburg I and Marburg IImutations.

[0007] German patent application 199 03 693.4 discloses a protease whichcan be isolated from blood plasma and which is able to activatecoagulation factor VII. This protease is also referred to as factorseven-activating protease (FSAP) (or as PHBP or PHBSP, corresponding toplasma hyaluronic acid-binding (serine) protease). FSAP therefore hasprocoagulant properties. A particular property of FSAP is that ofactivating single-chain plasminogen activators, such as prourokinase orsingle-chain tissue plasminogen activator (sct-PA). However, alone or incombination with plasminogen activators, FSAP can also be usedcorrespondingly to assist fibrinolysis, for example, in cases ofthrombotic complications.

[0008] The test systems which are now available and are described in theGerman patent applications 199 03 693.4 and 199 26 531.3 make itpossible not only to detect FSAP but also to quantify the FSAP antigencontent and determine the activity thereof in plasma. The antigendetermination is preferably carried out by means of an ELISA test. Onthe other hand, the activity can in principle be determined throughactivation of prourokinase to urokinase and conversion of a chromogenicsubstrate with subsequent extinction difference measurement.

[0009] German patent application 100 52 319.6 discloses the use of thesetest systems in investigations on healthy blood donors in which 5 to 10%of subjects were identified as having an average FSAP antigen contentbut a markedly reduced potential for activation of prourokinase. Sincethis probably also applied to the isolated, individual proteases, thecorresponding DNAs were analyzed for further investigation from bloodcells. It was surprisingly possible in this case to identify inparticular a mutation (single nucleotide polymorphism; SNP; G/A inposition 1601). This modification leads in the protein to a Gly to Gluamino acid exchange in position 511 of the mature protein or in aminoacid position 534 of the FSAP proenzyme including the signal peptide.This amino acid exchange results in FSAP losing the ability to activateprourokinase to urokinase or at least suffering a considerablediminution in activity. The aforementioned mutation, called FSAP MarburgI, has to date been found in all samples having an average antigencontent but a reduced activity in the formation of urokinase fromprourokinase. These results are described, for example, in U.S.application Ser. No. 09/912,559, to which this application claimspriority, and which is incorporated herein by reference.

[0010] For example, genomic DNA from the blood of two subjects withreduced activity and from four subjects with normal prourokinaseactivity was isolated, all exons amplified and then the FSAP DNAsequence was determined using the PCR primers. The result is shown inTable 1. A total of 4 nucleotide positions in the coding region werepolymorphic, i.e. at these positions two bases were detectedsimultaneously. It can therefore be assumed that these cases areheterozygous, having one wild type and one mutant allele. Two of these(at positions 183 and 957) are third base exchanges that do not resultin amino acid exchange. The other two, which were found only in the DNAof the subjects with reduced prourokinase activity, lead to amino acidexchanges as depicted in Table 1. TABLE 1 DNA sequence at nucleotidepositions* Subject No. ProUK activity 183 957 1177 1601 S83182 T G G G9689 normal T/C G G G 9690 normal T/C G G G 9704 normal T G G G 9706normal T G G G 9714 reduced T G G/C G/A 9715 reduced T G G/C G/A Aminoacid at position* Subject NT*:183 NT:957 NT:1177 NT:1601 No. ProUKactivity AA*:61 AA:319 AA:393 AA:534 S83182 His Lys Glu Gly 9689 normalHis Lys Glu Gly 9690 normal His Lys Glu Gly 9704 normal His Lys Glu Gly9706 normal His Lys Glu Gly 9714 reduced His Lys Glu/Gln Gly/Glu 9715reduced His Lys Glu/Gln Gly/Glu

[0011] In order to study the correlation of the two FSAP mutations withReduced prourokinase activating potency, the DNA of further individualswas Sequenced at these positions. The result is summarized in Table 2.All 6 subjects Having reduced prourokinase activating potency wereheterozygous at the nucleotide Position 1601 (Gly-Glu exchange at aminoacid 534), and four were additionally heterozygous at nucleotideposition 1177 (Glu-Gln exchange at amino acid 393). None of the 11subjects in total having normal prourokinase activating potency orprourokinase activating potency in the lower normal range had theabovementioned heterozygosities. This result suggests that the exchangein amino acid position 534 is causally linked to reduced prourokinaseactivity. TABLE 2 DNA sequence at Subject nucleotide position No. FSAPantigen ProUK activity 1177 1601 9714 Normal Low C/G A/G 9715 Normal LowC/G A/G 9802 Normal Low C/G A/G 10032 Normal Low G A/G 10039 Normal LowC/G A/G 10047 Normal Low G A/G 9698 Lower normal range Lower normalrange G G 9702 Lower normal range Lower normal range G G 9711 Lowernormal range Lower normal range G G 9712 Lower normal range Lower normalrange G G 10038 Lower normal range Lower normal range G G 9689 NormalNormal G G 9690 Normal Normal G G 9704 Normal Normal G G 9706 NormalNormal G G 9803 Normal Normal G G 10043 Normal Normal G G

[0012] Some embodiments of this invention therefore relate to anatherothrombosis risk factor that consists of a mutant of coagulationfactor VII-activating protease (FSAP). In some embodiments of thisinvention, the risk factor is a mutant in which the FSAP proenzyme,including the signal peptide, has a Gly/Glu exchange at amino acidposition 534. This mutation is herein called the Marburg I

[0013] The corresponding nucleotide sequence of the FSAP proenzymeincluding the signal peptide shows a G/A base exchange at nucleotideposition 1601. The sequences of the above FSAP species are provided inTable 3.

[0014] The Marburg I mutation is sometimes found together with a Glu/Glnexchange at position 393 (position 370 in the mature FSAP proteinwithout the leader sequence), resulting from a G to C mutation atposition 1177 in the nucleotide sequence corresponding to the FSAPproenzyme including the signal peptide. The Glu/Gln exchange at position393 is herein called the Marburg II mutation. TABLE 3 NT:1177 NT:1601SEQ ID NO. Description AA:393 AA:534 1 wild-type nucleotide G G 2Marburg I nucleotide G A 3 Marburg II nucleotide C G 4 Marburg I and IIA nucleotide 5 wild-type protein Glu Gly 6 Marburg I protein Glu Glu 7Marburg II protein Gln Gly 8 Marburg I and II protein Gln Glu

[0015] A PCR test for the Marburg I and II mutations was established andused to investigate the DNA of the subjects recruited for the Bruneckstudy. The FSAP Marburg I mutation was found in 4.5% of all the samplesanalyzed. These findings were then assessed using the individual datacollected during the study to assess the development and progression ofatherosclerosis.

[0016] Surprisingly, the Marburg I mutation correlates with an increasedrisk of developing arterial atherosclerosis. An odds ratio of about“6.6” was calculated for this FSAP mutation, i.e. a risk on the arterialside that is comparable with the risk of APC resistance in the venousregion. In this study the Marburg I polymorphism was the risk factorwith the highest odds ratio of all factors investigated, as shown intable 4. It is particularly surprising that this mutation represents anindependent risk factor, making its own, marked contribution to thedevelopment and progression of atherosclerosis after allowance for allpreviously known risk factors. This realization and the determination ofthe sequences corresponding to the FSAP Marburg I mutation may thereforeimprove the prospects of diagnosing and treating heart diseases andvascular disorders caused by atherosclerosis.

[0017] Atherothrombosis frequently leads, for example, to coronaryartery disease followed by myocardial infarctions. Depending on thevessels affected, the organ supplied thereby becomes involved. In thecase of the carotid artery, this results in the brain beingundersupplied with nutrients and oxygen and may, in the worst case, leadto a stroke. Other organs affected by atherothrombosis and subject tothe risk of vascular occlusive disease and the sequelae resultingthereform are also affected by the FSAP Marburg I mutant. This mayresult, for example, in disorders of the kidneys, liver, lungs, andother disorders.

[0018] Thus, the risk factor of this invention may indicate a geneticpredisposition to arterial thrombosis, and/or a genetic predispositionto the development of thromboses. The risk factor may also indicate agenetic predisposition to the development of atherosclerotic disordersand their sequelae, such as coronary artery disease, acute myocardialinfarction, pulmonary embolism, peripheral artery occlusion, acuteischemic stroke, and thromboembolism, in addition to arterialthrombosis. The risk factor may also indicate a genetic predispositionto thrombotic disorders and their sequelae, such as arterial and venousthrombosis, deep vein thrombosis, acute myocardial infarction, pulmonaryembolism, peripheral artery occlusion, acute ischemic stroke, andthromboembolism. The risk factor may also indicate a geneticpredisposition to at least one of arterial and venous occlusivedisorders, to at least one of atherosclerotic and thromboticrestrictions of organ functions, as well as to one or more of anginapectoris, myocardial infarction, and strokes.

[0019] Methods for examining the structure, sequence, and activity ofthe FSAP Marburg I and II mutants are described in the aforementionedGerman patent applications, in particular in German patent application1090 52 319.6, as well as in U.S. application Ser. No. 09/912,559, allof which are incorporated herein by reference. These methods includemeasurement of the FSAP protease activity, preferably in combinationwith an FSAP antigen test, and determination of the nucleotide sequencein the mutated region by suitable test systems.

[0020] The arterial thrombosis risk factor of the invention may thus bedefined as one or more FSAP mutants that have lost the ability toactivate single-chain plasminogen activators or for which this abilityis at least impaired. In some embodiments, the risk factor ischaracterized by an FSAP mutant that has lost the ability to activateprourokinase or for which this ability is reduced.

[0021] A genetic predisposition to the development of arterialthrombosis can thus be identified by detecting one or more of theaforementioned FSAP mutants. Detection, as used herein, meansdetermining if a mutant FSAP is present in a sample. Detection may becarried out by a variety of methods, as described below.

[0022] Detection of these FSAP mutants also indicates the predispositionto the development of arterial thromboses and the predisposition to thedevelopment of atherosclerotic or thrombotic disorders and theirsequelae, such as arterial and venous occlusive disorders, coronaryartery disease, acute myocardial infarction, pulmonary embolism,peripheral artery occlusion, acute ischemic stroke, deep veinthrombosis, and thromboembolism. The predisposition to development ofatherosclerotic or thrombotic restrictions of organ functions is afrequent cause of angina pectoris, myocardial infarction or strokes. Itis typical of all cases that the potential for activation ofsingle-chain plasminogen activators, such as single-chain tissueplasminogen activator (sc-tPA), and single-chain urinary plasminogenactivator (sc-uPA), or prourokinase, is reduced. The reduction of thisactivation potential can be detected in the blood but especially in theplasma.

[0023] In view of the great importance of FSAP mutants asatherothrombosis or arterial thrombosis risk factors, diagnostic methodsfor detecting them are very important. They may be based on determiningat least one of a reduced FSAP antigen concentration and a reducedactivity of FSAP in the body fluids of an individual. This may entaildetermination of the potential for activation of single-chainplasminogen activators, such as prourokinase, in the body fluids.

[0024] In the context of the present invention, an individual, or donor,may be a mammal, such as a human. Relevant body fluids include wholeblood, blood plasma, serum, as well as lymphatic, cerebrospinal,pleural, pericardial, peritoneal, and synovial fluids, tears, seminalplasma, and cell lysates.

[0025] Some embodiments of the invention include detecting heterozygousor homozygous mutants of the FSAP proenzyme gene with a G/A baseexchange at nucleotide position 1601 by analysing the genomic DNA of anindividual, or the mRNA or cDNA derived therefrom. Some embodiments ofthe invention include detecting heterozygous or homozygous mutants ofthe FSAP proenzyme gene with a G/C base exchange at position 1177. Insome embodiments, both of these base exchanges may be detected.

[0026] In other embodiments, FSAP mutants may be detected at the proteinlevel. Specific monoclonal or polyclonal antibodies may be used for thispurpose, as well as their corresponding Fab or F(ab′)₂ fragments.Histological investigation methods on tissues or in solutions extractedfrom tissues are also available. For examples of relevant tissues in thecontext of this invention, see table 4 below.

[0027] Exemplary antibodies are those specific for one or more ofwild-type FSAP, including its proenzyme with or without the signalsequence, and its fragments; and FSAP mutants comprising at least one ofa Glu to Gln exchange at amino acid position 393 and a Gly to Gluexchange at amino acid position 534, as well as their proenzymes with orwithout the signal sequence, and their fragments. Antibodies herein thatspecifically recognize both wild-type and mutant FSAP sequences, such assequences corresponding to full-length active enzymes, proenzymes andenzyme fragments, are termed “FSAP-specific,” while those that arespecific for only wild-type or only mutant FSAP sequences are termed“wild-type FSAP-specific” and “mutant FSAP-specific,” respectively.

[0028] In some embodiments, the diagnostic method includes incubating asample that might contain FSAP mutant(s) with a first antibodyimmobilized on a solid support, and, after washing, adding a second,labelled antibody, washing again and measuring the signal elicited bythe second antibody, wherein the second, labelled antibody may be awild-type FSAP-specific antibody.

[0029] Another method comprises incubating a sample that might containFSAP mutant(s) with a first, wild-type FSAP-specific antibody,immobilized on a solid support, and, after washing, adding a second,labelled antibody, washing again and measuring the signal elicited bythe second antibody.

[0030] A further method comprises immobilizing the sample that mightcontain FSAP mutant(s) on a support, adding a labelled antibody, aloneor mixed with an unlabelled antibody, and detecting the labelledantibody.

[0031] Yet another method comprises mixing an antibody immobilized on asupport with the sample that might contain FSAP mutant(s) in thepresence of a labelled FSAP mutant, and measuring the signal elicited bythe label.

[0032] In the methods described above, the first or second antibodies,unless stated otherwise, may include FSAP-specific, wild-typeFSAP-specific, or mutant FSAP-specific antibodies.

[0033] An example diagnostic method in which the activity of FSAP ismeasured comprises incubating an FSAP-containing sample on a solidsupport onto which an FSAP-specific antibody has previously beencoupled, and then, after washing out the free support, incubating theFSAP immobilized thereon with reagents that allow determination of FSAPactivity.

[0034] Some embodiments include diagnostic methods in which antibodiesare used to detect FSAP mutants by Western blotting for immunohistology,fluorescence-activated cell sorting (FACS), or comparable methods.

[0035] The diagnostic methods of the invention may also be carried outby the ELISA technique. This entails binding an FSAP and/or FSAP mutantto a matrix, for example to a microtiter plate. For optimal presentationof the FSAP and/or FSAP mutant, it is possible to coat the plate withmonoclonal or polyclonal antibodies, or the F(ab′)₂ or Fab fragmentsthereof, before loading with FSAP and/or FSAP mutants. Since FSAP andFSAP mutants generally bind very well to dextran sulfate, heparin andsimilar substances, coating with these agents is also possible prior toloading with FSAP and/or FSAP mutants. After the support or microtiterplate has been washed, it is blocked where appropriate with the agentsknown for this purpose, such as detergent or albumin, washed and thenincubated with the solution to be tested. The solutions containingFSAP-specific antibodies include blood serum, plasma, and other bodyfluids, as well as synovial fluids, cerebrospinal fluid, saliva, tears,seminal plasma or cell lysates.

[0036] Incubation and washing of the support is followed by use of asuitable detection reagent. The test substances required for detectingthe various antibody classes, such as IgG, IgM, IgA, IgE, and therelevant subclasses, are commercially available as labelled reagents.Detection and quantification of the antibody titer can then take placeby a photometric examination, for example, by measurement of theextinction brought about by cleavage of a chromogenic substrate by anenzyme coupled to the anti-human antibody. It is also possible tomeasure the fluorescence emitted by a fluorescent group connected to theantibody used for the detection. In addition, it is possible to carryout the detection with a radiometric measurement, if the substance usedfor detection is labelled with a radioactive group. Diagnostic methodsin which the bound human antibodies are incubated with a labelledanti-human immunoglobulin or fragments thereof, or labelled protein A orprotein G, and in which the signal emitted by the bound, labelledsubstance is determined, have already proved very suitable in manyinstances.

[0037] It is also possible to detect the antibodies by a photometricmeasurement of the extinction caused by cleavage of a suitablechromogenic or fluorogenic substrate by enzyme-coupled anti-humanantibodies or fragments thereof, or protein A or protein G. Diagnosticmethods in which antibodies are detected by measuring the fluorescencecaused by a bound substance labelled with fluorescent groups are alsosuitable.

[0038] In some embodiments, monoclonal antibodies were prepared andcharacterized as follows:

Immunization

[0039] Three female balb/c mice (approx. 6 weeks old) were immunizedwith FSAP. The first injection consisted of 0.2 ml of the antigen (10μg) mixed with 0.2 ml of complete Freund's adjuvant. In the threefollowing boost injections (each 2 weeks apart) the antigen (20 μg in0.2 ml) was administered without adjuvant (all injections i.p.). Theimmunogen was diluted in PBS. After the last injection, the serum titerwas determined by means of indirect ELISA by coating a microtiter platewith FSAP. The mouse with the highest serum titer was selected for thefusion.

Fusion

[0040] About three weeks after the last application, the antigen wasadministered on three successive days (10 μg in 0.1 ml i.v.). On thenext day (day 4) the mouse was sacrificed after taking blood. The spleenwas removed and the spleen cells were isolated. The spleen cells werethen fused with the murine myeloma cell line SP2/0-Ag 14. The fusionreagent was polyethylene glycol 4000 (Merck). The fusion was carried outusing a modification of the original Köhler/Milstein method. The cellswere distributed on 24-well culture plates. The medium used was Dulbeccomod. Eagle's medium with 10% fetal calf serum and HAT for selection.After about two weeks, the cell clones grown were transferred to thewells of a 48 well plate and coded.

Hybridoma Screening

[0041] The culture supernatant was taken from 1728 grown clones andassayed by means of ELISA for the presence of mouse IgG. With the aid ofimmobilized FSAP, mouse IgG-positive supernatants were tested forspecificity (ELISA). Of the cell lines assayed, 108 cell lines wereidentified as specific for FVII activator and stored in the frozenstate.

[0042] The two hybridoma cell lines denoted DSM ACC2453 and DSM ACC2454were selected for further studies. These cell lines were deposited onApr. 5, 2000, with the DSMZ—Deutsche Sammlung Von Mikroorganismen undZellkulturen GmbH, a depository which is subject to the Treaty ofBudapest regulations. The specificity of the antibodies produced by saidcell lines was confirmed by BIACORE and binding kinetics weredetermined. The two monoclonal antibodies are of the IgG1 type.

[0043] With the aid of the described antibodies against FSAP wild typeand against its mutants, it is possible to carry out diagnostic methodsfor detecting the mutants by:

[0044] a) incubating a sample that could contain one or more FSAPmutants with a first antibody, immobilized on a solid support, then,after washing, adding a second, labeled antibody and, after washing outagain, measuring the signal produced by the second antibody, wherein thesecond antibody may comprise a wild-type FSAP-specific antibody; or

[0045] b) incubating a sample that could contain one or more FSAPmutants with a first antibody immobilized on a solid support, then,after washing, adding a second, labeled antibody and, after washing outagain, measuring the signal produced by the second antibody, wherein thefirst antibody is a wild-type FSAP-specific antibody; or

[0046] c) immobilizing a sample that could contain one or more FSAPmutants on a support and detecting the sample with a labeled antibody,alone or in a mixture with an unlabelled antibody; or

[0047] d) incubating a sample that could contain one or more FSAPmutants with an antibody immobilized on a support in the presence of alabeled FSAP mutant, and measuring the signal produced by the label.

[0048] Antibody fragments, such as Fab and F(ab′)₂ fragments, may alsobe used in the diagnostic methods in some embodiments.

[0049] An example diagnostic method in which the FSAP activity ismeasured may include incubating the protease-containing sample with asolid support to which at least one of an FSAP-specific antibody, awild-type FSAP-specific antibody, or a mutant FSAP-specific antibody hasbeen coupled beforehand and, after washing out the solid support,incubating the FSAP fixed to the support with reagents which allowdetermination of its activity.

[0050] In this connection, FSAP activity, such as protease activity, canbe measured by photometric determination of the extinction appearingfollowing the action on chromogenic substrates.

[0051] It is also possible to determine FSAP protease activity bymeasuring:

[0052] its action of inactivating blood clotting factors VIII/VIIIa orV/Va or

[0053] its action of shortening blood clotting times in global clottingassays or

[0054] its action of activating plasminogen activators or

[0055] its action of activating blood clotting factor VII.

[0056] In some embodiments, FSAP action of activating plasminogenactivators may be measured, by examining FSAP activation of the

[0057] single-chain urokinase (scuPA, single chain urokinase plasminogenactivator) or the

[0058] single-chain-tPA (sctPA, single chain tissue plasminogenactivator).

[0059] The mutations responsible for the reduction of prourokinaseactivating potency can be detected at the DNA and RNA level by usingmethods that are also used for detecting single nucleotidepolymorphisms, for example

[0060] cDNA amplification of mRNA or amplification of the genomic DNAand sequencing;

[0061] detection of the mutation at the cDNA level or genomic DNA levelor their amplification by

[0062] hybridization with sequence-specific probes which may also carrylabels for the detection, such as enzymes, alkaline phosphatase, HRP andtheir substrates, fluorescent dyes, also reporter-quencher pairs (suchas, for example, scorpions, molecular beacons, TaqMan probes),radioisotopes, chromophores, chemiluminescence labels andelectrochemiluminescence labels) or

[0063] methods such as selective 2′-amine acylation, electrochemicaloxidation of nucleic acids by “minor groove binder” oligonucleotideconjugates, or by HPLC.

[0064] On the basis of the test results which were obtained by theabovementioned antigen assays and activity assays it was possible tostudy three groups of healthy donors regarding potential mutations atthe genomic level. For this purpose, blood was taken from the donors andthe blood cells were separated from the plasma by centrifugation. Theplasmas were then used to quantify the FSAP antigen and activity levelsand were divided according to the latter into three groups, namely into“normal/normal,” “lower normal range/lower normal range” and“normal/low.” The blood cells obtained were then used to extract genomicDNA and determine the FSAP genotype. The results depicted above in Table2 were determined.

[0065] Based on these results, it is now possible to detect rapidly oneor both of the mutants described, whether their genotype is heterozygousor homozygous, at the level of the corresponding FSAP nucleotidesequence. Whereas the abovementioned antigen and activity assaysreflected quite well the genotype in a healthy donor, this can becomedifficult or impossible when the FSAP plasma levels are influenced.Thus, parameters such as hormonal fluctuations, lifestyle, etc., as wellas pathological conditions, may strongly influence antigen and/oractivity levels. As described in the German Patent application 199 26531.3, the measurable FSAP activity during a heart attack can increasemarkedly compared with the normal value with scarcely increased antigencontent. As a result, donors that have a reduced FSAP activity whenhealthy, now appear to be “average.”

[0066] For example, studies on whether patients with FSAP mutations runan increased risk of suffering thrombotic complications such as heartattacks are possible only with difficulty, owing to the abovementionedrestrictions. On the other hand, for example, liver failures may lead toreduced plasma levels, and this likewise may lead to misinterpretationsof the “true” genetic predisposition. In contrast, a FSAP mutation assayat the DNA and/or RNA level is independent of temporary events. Thecombination of all of the assays mentioned allows a complete picture ofthe donor/patient, i.e. the evaluation of a potential mutation and ofthe acute state regarding an influence on the antigen-activity ratio.This may result in prophylactic and therapeutic measures.

[0067] As described above, heterozygous individuals whose blood plasmacontains normal FSAP at about 50% and the FSAP mutant at about 50% havebeen found. This results in an about 50% reduced activity level ofplasmas in which both types of FSAP molecules are present. A very smallproportion of individuals was found to be homozygous, their blood plasmacontaining the FSAP mutant at 100%, in which the prourokinase activationpotency was virtually abolished. Plasma pools which have been obtainedfrom the blood of 100 and more donors therefore also contain 5 to 10% ofFSAP mutants, depending on the population. This results in acorresponding probability to receive, in blood transfusions, donor bloodplasma that contains an FSAP mutant. If blood containing an FSAP mutantis administered to a recipient who cannot produce the mutant, the mutantmay be recognized as extraneous and appropriate antibodies can begenerated. Subsequent administration of the FSAP mutant at a later stagemay lead to immunological reactions in the recipient, the side effectsof which are familiar to the skilled worker.

[0068] Conversely, in a homozygous blood recipient who produces only anFSAP mutant but not normal FSAP, the latter is recognized as“extraneous” and the appropriate antibodies against it are produced.

[0069] FSAP affects hemostasis and the cellular processes connectedtherewith. By involvement in blood clotting and/or fibrinolysis, it alsoaffects the wound healing reaction. Moreover, FSAP, due to its propertyof having a high affinity to glycosaminoglycans, can bind to cells andother matrices and therefore is probably physiologically andpathophysiologically involved in cell migration and cellular-proteolyticprocesses.

[0070] FSAP-specific antibodies thus may influence all FSAP-mediatedactivities. In the case of autoantibodies against FSAP appearing, it ispossible that, in addition to an impairment of the physiologicalfunctions, immunocomplexes (FSAP+antibody) contribute to side effects ofknown autoimmune diseases. This may lead, for example, to vasculitideslocally in the endothelium. Neutralization of FSAP activity as aprofibrinolytic agent could also contribute to a thrombosis-promotingstate.

[0071] There is, therefore, a need for a diagnostic method for detectingthe above described antibodies.

[0072] Some embodiments of the invention, therefore, relate todiagnostic methods for detecting antibodies against factorVII-activating protease (FSAP) and/or against one or more FSAP mutantsformed by the exchange of one or more amino acids. The method maycomprise mixing a sample which could contain antibodies reactive withthe FSAP and/or FSAP mutants fixed to a solid support, incubating, and,after washing, detecting the antibody bound to the FSAP(s) with alabeled human anti-immunoglobulin or a labeled protein A and determiningthe signal emitted by the bound labeled substance.

[0073] This diagnostic method may also be carried out using the ELISAtechnique in which FSAP and/or one or more FSAP mutants are bound to amatrix, for example, to a microtiter plate. For optimal presentation ofthe FSAP and/or FSAP mutants, the plate may be coated beforehand withmonoclonal or polyclonal antibodies or their F(ab′)₂ or Fab fragmentsprior to loading the plate with the FSAP and/or FSAP mutants. Since FSAPand its mutants generally bind very well to dextran sulfate, heparin andsimilar substances, prior coating with these agents before FSAP bindingis also possible. After washing, the support or the microtiter plate mayadditionally be blocked and washed using agents known for this purpose,such as detergent or albumin, and then incubated with the solution to beassayed. FSAP antibody-containing solutions may include blood serum,plasma and other body fluids, such as synovial fluids, CSF, sputum,tears, and seminal plasma, as well as cell lysates.

[0074] After incubating and washing the support, a suitable detectionagent is then used. The assay substances necessary for detecting thevarious antibody classes such as IgG, IgM, IgA, IgE and the subclassesbelonging thereto, are commercially available as labeled reagents. Theantibody titer may be detected and quantified by a photometricdetermination measuring the extinction caused by cleavage of achromogenic substrate by an enzyme coupled to the anti-human antibody.It is also possible to measure fluorescence emitted by a fluorescentgroup linked to an antibody used for detection. It is also possible tocarry out the detection using radiometric measurement, if the substanceused for detection is labeled with a radioactive group.

[0075] The determination of antibodies against FSAP and/or in particularFSAP mutants makes it possible to identify the risk involved in a bloodtransfusion prior to carrying out the transfusion and to avoid dangerouscomplications by suitable measures.

[0076] Some embodiments of the invention relate to a diagnostic methodfor immunohistochemical detection of the blood clotting factorVII-activating protease (FSAP), its proenzyme, its mutants, or itsfragments. The method may comprise letting an FSAP-specific, labeled,monoclonal or polyclonal antibody, or one of its fragments, react with atissue sample, washing out the unbound antibody or its fragments, anddetermining the signal emitted from the bound antibody or one of itsfragments.

[0077] The method may also be carried out by letting an unlabelledmonoclonal or polyclonal antibody or antibody fragment, directed againstFSAP, its proenzyme, its mutants, or its fragments, react with a tissuesample, washing out the unbound antibody or its fragments, then lettinga labeled anti-antibody or its fragments react with the tissue sample,and, after washing out the unbound labeled anti-antibody, determiningthe signal emitted from the bound anti-antibody or its fragments.

[0078] It was also found that monoclonal or polyclonal antibodiesdirected against FSAP are very well suited to detecting FSAP in tissuesections of human origin, when the antibodies are labeled withchromophoric or luminescent groups. FSAP-specific polyclonal antibodiesobtained by immunization of rabbits, sheep, goats, or other mammals aresuitable for the detection as well as monoclonal antibodies.Particularly suitable for the histological specific detection of FSAPwhich may be present both in the active form and in the proenzyme form,as well as in a fragment, are the monoclonal antibodies of hybridomacell lines DSM ACC 2453 and DSM ACC 2454. Complexes of activated FSAPwith inhibitors such as antiplasmin may also be detected in this way.Suitable for this purpose are all common histological detection methodssuch as light microscopy, fluorescence microscopy and electronmicroscopy.

[0079] Suitable for detecting FSAP in the abovementioned methods areboth the complete polyclonal and monoclonal antibodies and theirfragments such as F(ab′)₂ or Fab, as long as they are labeled with adetectable group. The abovementioned antibodies or their fragments maybe applied alone or as a mixture. This is particularly recommended incase one of the recognized epitopes is obscured. For example, a proteindomain may not be accessible for an antibody due to cellularassociation, but is bound by another antibody having specificity for adifferent FSAP region. Antibodies which are directed against human FSAP,such as against one or more of the wild type and mutants of human FSAP,and which are described in more detail in the German Patent application100 52 319.6 may also be employed for detection of FSAP in tissuesections of human origin.

[0080] The findings obtained so far on the immunohistochemical detectionof FSAP can be summarized as follows:

[0081] FSAP is detected in almost all of the human tissues studied upuntil now;

[0082] endocrinologically active cells such as Leydig cells or theendocrinologically active cells of the islets of Langerhans of thepancreas are very strongly stained intracytoplasmatically usingantibodies against FSAP carrying chromophoric groups;

[0083] epithelia and endothelia display according to their location amore or less strong intracytoplasmatic immunoreaction with antibodiesagainst FSAP;

[0084] gangliocytes and dendrites of the cortex display highconcentrations of FSAP, and this is detected by a strongimmunohistological color reaction with chromophoric antibodies;

[0085] plasma cells display an intensive intracytoplasmatic colorationwith chromophoric antibodies against FSAP;

[0086] mesenchymal stroma cells display in complex tissues only a weakor no color reaction toward FSAP.

[0087] FSAP is thus a protein that can be regarded as a normal cellconstituent. So far FSAP was found located both intracellularly andextracellularly, with the former compartment being markedly morestainable. The inventive detection of FSAP by the mentioned antibodiesor their fragments makes it possible to identify the followingpathological processes:

[0088] endocrinologically active tumors and neuro-endocrine tumors;

[0089] angiogenic endothelia and endothelia of the capillaryendothelium; and also

[0090] angiogenically active tumors such as gliomas and glioblastomas,but also, for example, vascular tumors such as hemangioendothelioma orhemangiopericytoma and angiosarcoma;

[0091] wound healing reactions, granulation tissue and collagenoses;

[0092] atherosclerotic, (micro)thrombosed and necrotic areas;

[0093] neurodegenerative disorders such as Alzheimer's disease,Parkinson's disease or as spongiform encephalitides, for example causedby prion proteins;

[0094] gammopathies and myelomas.

[0095] FSAP may be detected by using monoclonal antibodies of hybridomacell lines DSM ACC 2453 or DSM ACC 2454.

[0096] The diagnostic method of the invention is illustrated in moredetail by the following example.

EXAMPLE I

[0097] The immunohistochemical reactivity of the FSAP-specificmonoclonal antibodies of hybridoma cell lines DSM ACC 2453 and DSM ACC2454 was studied by preparing from adult human tissue and malignanturological tumors 10 μm thick paraffin sections and subsequentlyde-waxing the sections which were treated in citrate buffer in themicrowave for 3 times 5 minutes. First, an unlabelled antibody of theabovementioned hybridoma cell lines was allowed to react with thesections for 30 minutes. After washing out the tissue section, a labeledanti-mouse detection antibody was allowed to react with the tissuelikewise for 30 minutes and then the bound FSAP antibody was madevisible by forming the APAAP complex (Alkaline Phosphatase/Anti-AlkalinePhosphatase complex) and by staining with chromogen and counterstainingwith hemalum.

[0098] As a negative control, each tissue was separately incubated withthe detection antibody—without prior incubation with the FSAPantibody—in order to make potential unspecific reactions of thedetection visible. In addition an antibody against α-keratin wasincluded as a positive control.

[0099] The results of the immunohistochemical study of normal humantissue are summarized in Table 4. TABLE 4 Antibodies against FSAP, cloneDSMZ ACC2454 and DSMZ ACC 2453 Human normal tissue 2454 2453 2454 2453Esophagus Appendix Squamous epithelium 2+ 2+ Epithelia 1+ 3+ Secretoryunits 0   0   Musculature 1+ 1+ Acinar ducts 2+ 2+ Lymphatic follicle 1+2+ Musculature 1+ 1+ Plasma cells 2+ 3+ Stroma 1+ 1+ − 2+ Vascularendothelium 1+ 1+ Vascular endothelium 2+ 2+ Pancreas Cardia (stomach)Epithelia 1+ 2+ Foveolar epithelium 0   0   Islets of Langerhans 3+ 1+Glandulae cardiacae 1+ 2+ Duct epithelium 2+ 2+ Mucous secretory units0   0   Vascular endothelium 1+ 1+ Oxyntic glands 3+ 3+ Salivary glandMusculature 1+ 1+ Mucous end units 0   0   Vascular endothelium 1+ 1+Serous end units 1+ 1+ − 2+ Corpus (stomach) Acinar ducts 1+ 1+ Foveolarepithelium 0   0 − 1+ Striate ducts 1+ 1+ Corpus gland body 2+ 2+Vascular endothelium 0 − 1+ 0 − 1+ Musculature 0   1+ Liver Vascuiarendothelium 1+ 1+ Hepatocytes 2+ 2+ Duodenum Bile ducts 0   0  Epithelia 0 − 1+ 1+ Vascular endothelium 1+ (1+) Brunner's glands 0  0   Gall bladder Musculature 0   0 − 1+ Epithelia 1+ 1+ Lymphaticfollicle 1+ 2+ Musculature 2+ 1+ Ganglion cells 2+ 3+ Vascularendothelium 2+ 1+ Vascular endothelium 1+ 1+ Cystic duct Small intestineEpithelium 3+ 3+ Epithelia 2+ 3+ Musculature 2+ 1+ Musculature 1+ 1+Ganglion cells 3+ 3+ Stroma 1+ 2+ Vascular endothelium 2+ 2+ Ganglioncells 3+ 3+ Testis Vascular endothelium 1+ 1+ Leydig cells 3+ 1+Colon/Rectum Sertoli cells 1+ − 2+ 1+ Epithelia 1+ 1+ Germ cells 1+ − 2+1+ Lymphatic follicles 1+ 1+ Vascular endothelium 1+ 1+ Plasma cells 1+1+ Rete testis Vascular endothelium 1+ 0   Epithelium 2+ 2+ EpididymisPlacenta Epididymis duct 2+ 2+ Chorionic epithelium 3+ 2+ Efferentductulus 2+ 2+ Amniotic epithelium 2+ 2+ Stroma 1+ 1+ Decidual cells2+−3+ 2+ Vascular endothelium 1+ 1+ Stroma cells 0   +/− Seminal glandVascular endothelium 1+ 1+ Epithelium 2+ 3+ Fetal membranes Musculature1+ 1+ Amniotic epithelium 3+ 2+ Vascular endothelium 2+ 2+ Decidualcells 3+ 1 + Deferent duct Fibroblasts 3+ Epithelium 2+ 3+ Cervix uteriLongitudinal muscle 0   +/− Glandular epithelium 0   0   layer Annularmuscle layer 2+ 3+ Vascular endothelium 1+ 0 − 1 Vascular endothelium 2+3+ Stroma 1+ 0 − 1 Prostate Fallopian tube Glandular epithelium 2+ 2+Epithelium 2+ 3+ Musculature 1+ 1+ Musculature 0   1+ Vascularendothelium 1+ − 2+ 1+ − 2+ Vascular endothelium 1+ 2+ Kidney BreastTubules 2+ 1+ Epithelia mammary 2+ 2+ gland lobules Glomerules 0   0  Duct epithelium 2+ 2+ secretory ducts Medullary epithelium 1+ 1+Fibroblasts 0   1+ Vascular endothelium 0 − 1+ 0 − 1+ Plasma cells 2+2+Bladder Vascular endothelium 1+ 0   Urothelium 2+ 1+ − 2+ ThyroidMusculature 2+ 1+ Follicular epithelium 2+ Plasma cells 2+ 2+ Stroma 1+1+ Fibroblasts 1+ − 2+ 1+ − 2+ Vascular endothelium 1+ 0   Peripheralnerve 0   Thymus Adrenal gland Hassall's bodies 2+ − 3+ 2+ Glomerularzone 2+ 1+ Follicles 1+ 2+ Fascicular zone 1+ − 2+ (1+) Mantle zone (1+)(1+) Reticular zone 3+ (1+) Starry sky 1+ 1+ macrophages Medulla 0   0  Spleen ++ Vascular endothelium 1+ (1+) Tonsils +/− +/− Endometrium Lymphnodes +/− +/− Glandular epithelium 3+ 2+ Maxillary sinus Stroma cells0   1+ Respiratory epithelium 2+ 2+ Myometrium 1+ 1+ Plasma cells 3+ 3+Vascular endothelium 1+ 2+ Vascular endothelium 1+ 1+ Lung Fatty tissue2+ 2+ Bronchial epithelium 2+ 1+ Vascular endothelium 2+ 2+ Alveolarepithelium 1+ − 2+ 1+ Skin Bronchial glands 1+ 1+ Epidermis 2+ 1+ − 2+Cartilage 3+ 1+ Dermis (1+) 0   Musculature 1+ 1+ Hypodermis (1+) 0  Alveolar macrophages 2+ 2+ Sweat glands 1 + 0   Elastic fibers 2+ − 3+2+ − 3+ Vascular endothelium 1+ 0   Vascular endothelium 1+ 1+Endocardium 0   0   Skeletal muscles 2+ 1+ Fibroblasts 2+ − 3+ 2+ − 3+

[0100] Endocrine cells such as the islets of Langerhans of the pancreas,the Leydig cells of the testicular interstitium, the decidual cells ofthe placenta, the oxyntic gland body of the stomach cardia, and thehighly cylindrical epithelium of the cystic duct, display a strongreaction, which in part, shows fine granules. Strongly positivereactions were observed in plasma cells located in tissue structures andganglionic cells and nerve cells of the cortex. The decidual cells, theamniotic epithelium and the fibroblasts of fetal membranes displayedvery strong immunohistological stainability, as did the epitheliumlining the seminal glands and the enterocytes of the small intestine.

[0101] Studies of formalin-fixed, paraffin-embedded tumor material ofurological tumors displayed a weak to moderately strongintracytoplasmatical reaction of different differentiatedadenocarcinomas of the prostate. Tumor cells of seminomatous testiculartumors showed only a weak intracytoplasmatic reaction whilenon-seminomatous tumors (embryonic carcinomas and chorionic carcinomas)had a widely increased stainability of the tumor cells, indicatingincreased concentrations of FSAP.

[0102] The diagnostic method of the invention thus allows animmunohistochemical detection of pathological processes in a widevariety of organs.

EXAMPLE II Bruneck Study Study Subjects

[0103] The Bruneck study is a prospective population study aimed atthrowing light on epidemiology and etiology of carotid atherosclerosis(1-6). The study population was recruited in 1990 as a sample stratifiedaccording to sex and age and all the Bruneck inhabitants from 40 to 79years of age (125 women and 125 men from each of decades 5 to 8 of age,n=1000). In total, 93.6% took part, with 919 completing the dataacquisition. During the follow-up period between summer 1990 and 1995(quinquennium₁—Q₁), a subgroup of 62 individuals died, while one subjectmoved house. Follow-up in the remaining population was 96.5% complete(n=826) (1-3). Before entry in the study, all the participants gavetheir consent after they had been informed about the study. As part ofthe follow-up in 1995, blood samples were taken to obtain DNA.Unsatisfactory PCR products were obtained in 16 cases, i.e. 810 men andwomen remained for the main analysis. Of these subjects, 94 died betweensummer 1995 and 2000 (quinquennium₂—Q₂). A total of 675 subjectsunderwent ultrasound investigation again in 2000 (follow-up rate amongthe survivors 94.3%) (6).

Clinical History and Examination

[0104] The study protocol included a clinical examination with priorityfor cardiological and neurological items and standardized questionnairesconcerning the current or past vulnerability due to potential vascularrisk factors (3-5). For smokers and former smokers, the average numberof cigarettes smoked each day, and the pack-years, were recorded. Thealcohol consumption was quantified as grams per day and classified infour categories (3). Systolic and diastolic blood pressures were themeans of three measurements, in each case measured after resting for ≧10minutes. Hypertension was defined as a blood pressure of ≧160/95 orintake of antihypertensive agents (WHO definition). A standardized oralglucose tolerance test was carried out on all subjects excepting thosepreviously known to be diabetic. Diabetes mellitus was entered aspresent for those subjects whose fasting blood glucose level was ≧140mg/dl (7.8 mmol) and/or who had a 2-hour level (oral glucose tolerancetest) of ≧200 mg/dl (11.0 mmol/l).

Laboratory Methods

[0105] After the subjects had taken no food and not smoked for at least12 hours, blood was taken from the antecubital vein (3-6). Totalcholesterol and cholesterol with high density lipoprotein weredetermined enzymatically (CHOD-PAP method, Merck, Darmstadt, Germany),and lipoprotein(a) concentrations were measured using an ELISA (Immuno,Vienna, Austria). The cholesterol with low density lipoprotein wascalculated from the Freidewald formula. Fibrinogen was measured by themethod of Clauss, and the antithrombin Ill using a chromogenic assay.The Leiden mutation of factor V was detected by allele-specific PCRamplification (3).

[0106] FSAP antigen concentrations and scuPA-activating effect weredetermined as described recently (7, 8). Stated briefly, an ELISA withmonoclonal antibodies (mAb) against FSAP was used for antigenquantification. The activity assay comprised an immunoadsorption ontomicrotiter plates coated with antibodies, a washing step and subsequentactivation of prourokinase by FSAP, which was quantified by photometricobservation of the amidolysis of a chromogenic substrate for urokinase.Pooled plasma from more than 200 healthy blood donors was used asarbitrary standard for both assays. A plasma equivalent unit (PEU) wasdefined as the FSAP antigenic activity present in one milliliter of thepooled plasma, which corresponds on average to 12 μg/ml (8).

[0107] DNA extraction and FSAP genotyping: high-quality DNA was obtainedfrom frozen whole blood using a GenomicPrep Blood DNA Isolation Kit(Amersham Pharmacia Biotech). Ten ml of extracted DNA were amplified in100 μl of 1×PCR standard reaction buffer with 50 pmol of thecorresponding exon-specific forward and reverse primer, 1.5 mM MgCl₂,0.2 mM dNTP and 2.5 units of Taq DNA polymerase (Perkin Elmer, Langen,Germany); an initial 2-minute denaturation at 94° C. was followed by 35thermocycles each for 30 seconds at 94° C., 30 seconds at 50° C. and 40seconds at 72° C., which was followed by a final elongation step at 72°C. for 5 minutes. The pairs of primers used have recently been describedin Roemisch J, Feussner A, Nerlich C, et al. The frequent Marburg Ipolymorphism impairs the prourokinase activating potency of the factorVII-activating protease. Blood Coag Fibrinol 2002; 13:1-9, which isincorporated herein by reference.

Scanning Protocol and Definition of the Ultrasound Endpoints

[0108] In the ultrasound examination, the internal carotid artery(bulbous and distal sections) and common carotid artery (proximal anddistal sections) on both sides were scanned using a 10 MHz probe and a 5MHz doppler (1,2). Atherosclerotic lesions were defined by twoultrasound criteria: 1) wall surface (protrusion into the lumen) and 2)wall texture (echogenicity). The maximum axial diameter of plaques wasdetermined in each of 16 vessel sections (intra-observation coefficientof variation 10% or 15% depending on the vessel section). The thicknessof the intima media was measured at the far walls of the common carotidartery (intra-observation coefficient of variation 7.9% (n=100)) (2).The scans were performed in 1990, 1995 and 2000 by the same experiencedultrasonic specialist, the clinical findings and laboratory values ofthe subjects being unknown to the ultrasonic specialist.

[0109] The development of atherosclerosis was characterized by theappearance of new plaques in previously normal sections. Thresholds of0.7 mm (common carotid artery) and 1.0 mm (internal carotid artery) wereintroduced as minimum requirements concerning the plaque diameters inthe definition of developing atherosclerosis, because smaller lesionswere difficult to distinguish from focal/diffuse wall thickenings (1).Progression of non-stenotic lesions was defined as a relativeenlargement of the plaque diameter of more than twice the measurementerror of the method (1). In the current analysis, both processes werecombined to a single result category referred to as “earlyatherogenesis” for easier presentation and because of the fact that mostof the described risk factors were common to these processes. An“advanced atherogenesis” was assumed whenever the criterion ofprogression was met and the lumen was narrowed by >40%. As describedelsewhere (1-5), the cutoff at 40% appeared to correspond to abiological threshold in our population, at which marked changes in thegrowth kinetics of plaques (continuous, slow and diffuse growth versusoccasional and focal expansions of prominent lesions), in the riskprofiles (conventional risk factors versus procoagulation risk factors)and in the process of vascular renewal (compensating or overcompensatingversus insufficient or even absent) occurred, indicating a switch in theunderlying pathogenetic mechanisms from conventional atherogenesis toatherothrombosis.

[0110] The reproducibility of the ultrasound categories was “nearlyperfect” (kappa coefficients of >0.8, obtained from two independentmeasurements carried out by the same ultrasound specialist in areproducibility sample of n=100 (1-3).

Statistical Analysis

[0111] Possible associations between FSAP mutations and the variousstages of atherogenesis were examined by means of logistic regressionanalysis. A base model was adjusted only in relation to age and sex.Multivariate equations were fitted by a stepwise progressive selectionprocedure as already described (p values for entry and exclusion 0.10and 0.15 respectively) (3, 10). Age and sex were additionally insertedinto these models in order to take account of the age and sex structureof the population sample. The main analysis was concentrated on theperiod between 1990 and 1995 (Q₁). Analysis of advanced atherogenesiswas restricted to subjects already suffering from atherosclerosis at thestart of the study (n=326).

[0112] The regression-standardized atherogenesis risks were calculatedfor a number of risk factors. The marginal method of the regressionadjustment procedure was used, because it is not based on therare-disease assumption (11).

Results

[0113] In the Bruneck study cohort (n=810), 36 subjects wereheterozygous for the Marburg I mutant of FSAP (17 men and 20 women) andone subject was homozygous, corresponding to an overall carrier rate[95% Cl] in the general population of 4.4% [3.0% to 5.8%]. Acosegregation of the Marburg II mutant (E393Q) of FSAP was observed in16 of the 37 individuals (43 percent, 8 men and 8 women), while theMarburg I mutant occurred in isolation in the remaining 21 subjects (57percent, 9 men and 12 women).

[0114] Plasma samples from the subpopulation (n=82) were investigatedfor FSAP antigen concentrations and correspondingprourokinase-activating effects. In 76 subjects with wild-type FSAP, theaverage (±2×SD) antigen concentrations, activity concentrations andactivity/antigen ratios were respectively 0.991 (0.552 to 1.430) PEU/ml,1.036 (0.614 to 1.458) PEU/ml and 1.07 (0.63 to 1.51). In contrastthereto, all six carriers of the Marburg I mutant in this subgroupshowed a distinctly reduced in vitro capacity to activate prourokinase(<0.150 to 0.626) and activity/antigen ratios of 0.38 to 0.58. There washardly any overlap in the distribution of these parameters in the twogenetic groups.

[0115] During the five-year follow-up period between 1990 and 1995 (Q₁),a total of 384 of the 810 subjects in the study (47.4%) developed newatherosclerotic lesions or showed extension of nonstenotic lesions(early atherogenesis), and 92 of 326 individuals (28.2%) withpre-existing plaques showed stenotic transformations (advancedatherogenesis). As expected, no relation was found between Marburg Imutant and early atherogenesis (age/sex adjusted, multivariate oddsratios [95% Cl] of 0.6 [0.3 to 1.4] and 0.7 [0.3 to 1.7]. Consistentwith this, there were no differences in the thickness of the intimamedia of the common carotid artery between the carriers of wild-typeFSAP (0.95 mm) and of the Marburg I mutant of FSAP (0.94 mm; P=0.853 forthe difference). However, it emerged that the mutant is a strong riskfactor for the advanced putative atherothrombotic stage in atherogenesis(age/sex adjusted odds ratio [95% Cl] 3.5 [1.1 to 11.4], P=0.036). Theassociation remained statistically significant on adjustment of thelogistic regression model for other relevant risk factors (tab. 1). Therisk profile for advanced stenotic atherosclerosis also includeddiabetes, a high fibrinogen concentration, a low antithrombinconcentration, a high platelet count, smoking, alcohol consumption(small amounts protective), Lp(a)>0.32 g/l and Leiden mutation of factorV. There were no sex-specific differences in the risk factors, and noevidence of differential effects of the Marburg I mutant was found insubpopulations arranged according to age, level of risk and life style.Exclusion of subjects taking aspirin, antihypertensive agents,antidiabetics or lipid-lowering agents likewise did not affect theresults. Regression-standardized risks of advanced atherogenesis for anumber of major risk factors (Marburg I mutant, IGT/diabetes, highlipoprotein(a) concentration, smoking, factor V mutation, highfibrinogen concentration and low antithrombin concentration) are shownin table 2. Subjects with none of the risk factors had a low risk fordevelopment/progression of carotid stenosis, whereas subjects with acluster of more than two factors almost obligatorily experiencedadvanced atherogenesis.

[0116] The Marburg II mutant had no effect on in vitro activation ofsingle-chain plasminogen activators by FSAP. Accordingly, it was notunexpected that no association between this mutation and atherogenesiscould be found in our analyses. On comparison of subjects with wild-typeFSAP and carriers of the Marburg II mutant, of the Marburg I mutant andcarriers of both genetic deviations, the multivariate odds ratios [95%Cl] for advanced atherosclerosis were 1.6 [02 to 13.7], P=0.669, 6.2[1.1 to 36.0], P=0.048 and 7.1 [1.1 to 45.1], P=0.037.

[0117] To demonstrate that our findings are also consistent over longerperiods, the calculations were repeated with the data from the ten-yearfollow-up period between 1990 and 2000 (Q₁₊₂). In these equations, themultivariate relation between the Marburg I mutant of FSAP and advancedatherosclerosis (same adjustments as for the original analysis) wasagain statistically significant (multivariate odds ratio [95% Cl] 4.1[1.1 to 14.8], P=0.045). TABLE 5 Multivariate logistic regressionanalysis of advanced atherogenesis according to age, sex, Marburg Imutant of FSAP and other potential vascular risk factors. Means ±standard deviation (%) AS − AS + Odds ratio Variable (n = 234) (n = 92)(95% CI) P value Step Age, y 64.9 ± 9.2 67.8 ± 8.0 1.87(1.19 − 2.92).0064 0 Female sex 109(46.6%) 32(34.8%) 0.56(0.25 − 1.25) .1555 0Glucose tolerance <.0001 1 IGT  20(8.5%) 16(17.4%) 3.31(1.37 − 7.99).0081 DM  10(8.1%) 21(22.8%) 6.38(2.71 − 14.99) <.0001 Cigarettes/day3.2 ± 7.2 6.6 ± 9.6 1.77(1.30 − 2.40) .0003 2 Lp(a) > 0.32 g/l 36(15.4%) 25(27.2%) 4.06(1.83 − 8.96) .0005 3 Alcohol consumption .00434    <1 g/d 114(48.7%) 42(45.6%) 1.00  1-50 g/d  60(25.7%) 15(16.3%)0.26(0.10 − 0.66) .0046 51-99 g/d  37(15.8%) 17(18.5%) 1.03(0.40 − 2.70).9475  ≧100 g/d  23(9.8%) 18(19.6%) 1.90(0.63 − 5.69) .2535 Fibrinogen,g/l 2.7 ± 0.6 2.9 ± 0.6 1.53(1.12 − 2.09) .0083 5 Marburg I FSAP 5(2.1%)  8(8.7%) 6.63(1.58 − 27.72) .0099 6 mutation Factor V mutation 5(2.1%)  7(7.6%) 4.70(1.19 − 18.55) .0291 7 Antithrombin III, % 96.3 ±13.0 92.8 ± 16.4 0.74(0.55 − 1.00) .0500 8 Platelet count, × 10⁹/l 217.4± 56.5  230.3 ± 56.6  1.32(0.98 − 1.77) .0769 9

[0118] Odds ratios (OR), 95% confidence interval (95% Cl) and p values(P) were derived from the logistic regression analysis of advancedatherosclerosis (development/progression of stenotic carotidatherosclerosis) in relation to age, sex and vascular risk factors. Themodel was fitted by a stepwise progressive selection process (step . . .entry step). The ORs were calculated for a 1−SD unit change of givenvariables.

[0119] AS−: group without advanced atherogenesis, AS+: group withadvanced atherogenesis. This analysis was concentrated on the 326subjects who already suffered from atherosclerosis at the start of thestudy in 1990.

REFERENCES

[0120] 1. Kiechl S, Willeit J. The natural course of atherosclerosis.Part I: incidence and progression. Arterioscler Thromb Vasc Biol 1999;19: 1480-90.

[0121] 2. Kiechl S, Willeit J. The natural course of atherosclerosis.Part II: vascular remodeling. Arterioscler Thromb Vasc Biol 1999; 19:1491-8.

[0122] 3. Willeit J, Kiechl S, Oberhollenzer F, et al. Distinct riskprofiles of early and advanced atherosclerosis. Prospective results fromthe Bruneck Study. Arterioscler Thromb Vasc Biol 2000; 20: 529-37.

[0123] 4. Kiechl S, Egger G, Mayr M, et al. Chronic infections and therisk of carotid atherosclerosis. Prospective results from a largepopulation study. Circulation 2001; 103: 1064-70.

[0124] 5. Kiechl S, Willeit J, Egger G, Poewe W, Oberhollenzer F. Bodyiron stores and the risk of carotid atherosclerosis. Prospective resultsfrom the Bruneck Study. Circulation 1997; 96: 3300-7.

[0125] 6. Kiechl S, Lorenz E, Reindl M, Wiedermann C J, Oberhollenzer F,Bonora E, Willeit J, Schwartz D A. Toll-like receptor 4 polymorphismsand atherogenesis in humans. N Engl J Med 2002; 347: 185-92.

[0126] 7. Roemisch J, Feussner A, Stohr H A. Quantification of thefactor VII- and single-chain plasminogen activator-activating proteasein plasmas of healthy subjects. Blood Coagul. Fibrinolysis. 2001; 12:375-83.

[0127] 8. Kannemeier C, Feussner A, Stohr H A, Weisse J, Preissner K T,Roemisch J. Factor VII and single-chain plasminogen activator-activatingprotease: activation and autoactivation of the proenzyme. Eur J Biochem.2001; 268: 3789-96.

[0128] 9. Roemisch J, Feussner A, Nerlich C, Stoehr H A, Weimer T. Thefrequent Marburg I polymorphism impairs the prourokinase activatingpotency of the factor VII-activating protease (FSAP). Blood CoagFibrinol 2002; 13: 1-9.

[0129] 10. Hosmer D W, Lemeshow S. Applied Logistic Regression. NewYork: John Wiley & Sons, 1988.

[0130] 11. Wilcosky T C, Chambless L E. A comparison of directadjustment and regression adjustment of epidemiological measures. J.Chron Dis 1985; 38: 849-56.

Figure Legend

[0131]FIG. 1 shows the regression-adjusted risk of advancedatherogenesis as a function of the vascular risk factors present in anindividual (Marburg I mutant of factor VII-activating protease,IGT/diabetes, lipoprotein(a) concentration >0.32 g/l, smoking, Leidenmutation of factor V, fibrinogen concentration (Q₅, >3.2 g/l) andantithrombin concentration (Q₁, <84%)).

1 8 1 1683 DNA Homo sapiens 1 atgtttgcca ggatgtctga tctccatgttctgctgttaa tggctctggt gggaaagaca 60 gcctgtgggt tctccctgat gtctttattggaaagcctgg acccagactg gacccctgac 120 cagtatgatt acagctacga ggattataatcaggaagaga acaccagtag cacacttacc 180 catgctgaga atcctgactg gtactacactgaggaccaag ctgatccatg ccagcccaac 240 ccctgtgaac acggtgggga ctgcctcgtccatgggagca ccttcacatg cagctgcctg 300 gctcctttct ctgggaataa gtgtcagaaagtgcaaaata cgtgcaagga caacccatgt 360 ggccggggcc aatgtctcat tacccagagtcctccctact accgctgtgt ctgtaaacac 420 ccttacacag gtcccagctg ctcccaagtggttcctgtat gcaggccaaa cccctgccag 480 aatggggcta cctgctcccg gcataagcggagatccaagt tcacctgtgc ctgtcccgac 540 cagttcaagg ggaaattctg tgaaataggttctgatgact gctatgttgg cgatggctac 600 tcttaccgag ggaaaatgaa taggacagtcaaccagcatg cgtgccttta ctggaactcc 660 cacctcctct tgcaggagaa ttacaacatgtttatggagg atgctgaaac ccatgggatt 720 ggggaacaca atttctgcag aaacccagatgcggacgaaa agccctggtg ctttattaaa 780 gttaccaatg acaaggtgaa atgggaatactgtgatgtct cagcctgctc agcccaggac 840 gttgcctacc cagaggaaag ccccactgagccatcaacca agcttccggg gtttgactcc 900 tgtggaaaga ctgagatagc agagaggaagatcaagagaa tctatggagg ctttaagagc 960 acggcgggca agcacccatg gcaggcgtccctccagtcct cgctgcctct gaccatctcc 1020 atgccccagg gccacttctg tggtggggcgctgatccacc cctgctgggt gctcactgct 1080 gcccactgca ccgacataaa aaccagacatctaaaggtgg tgctagggga ccaggacctg 1140 aagaaagaag aatttcatga gcagagctttagggtggaga agatattcaa gtacagccac 1200 tacaatgaaa gagatgagat tccccacaatgatattgcat tgctcaagtt aaagccagtg 1260 gatggtcact gtgctctaga atccaaatacgtgaagactg tgtgcttgcc tgatgggtcc 1320 tttccctctg ggagtgagtg ccacatctctggctggggtg ttacagaaac aggaaaaggg 1380 tcccgccagc tcctggatgc caaagtcaagctgattgcca acactttgtg caactcccgc 1440 caactctatg accacatgat tgatgacagtatgatctgtg caggaaatct tcagaaacct 1500 gggcaagaca cctgccaggg tgactctggaggccccctga cctgtgagaa ggacggcacc 1560 tactacgtct atgggatagt gagctggggcctggagtgtg ggaagaggcc aggggtctac 1620 acccaagtta ccaaattcct gaattggatcaaagccacca tcaaaagtga aagtggcttc 1680 taa 1683 2 1683 DNA Homo sapiens 2atgtttgcca ggatgtctga tctccatgtt ctgctgttaa tggctctggt gggaaagaca 60gcctgtgggt tctccctgat gtctttattg gaaagcctgg acccagactg gacccctgac 120cagtatgatt acagctacga ggattataat caggaagaga acaccagtag cacacttacc 180catgctgaga atcctgactg gtactacact gaggaccaag ctgatccatg ccagcccaac 240ccctgtgaac acggtgggga ctgcctcgtc catgggagca ccttcacatg cagctgcctg 300gctcctttct ctgggaataa gtgtcagaaa gtgcaaaata cgtgcaagga caacccatgt 360ggccggggcc aatgtctcat tacccagagt cctccctact accgctgtgt ctgtaaacac 420ccttacacag gtcccagctg ctcccaagtg gttcctgtat gcaggccaaa cccctgccag 480aatggggcta cctgctcccg gcataagcgg agatccaagt tcacctgtgc ctgtcccgac 540cagttcaagg ggaaattctg tgaaataggt tctgatgact gctatgttgg cgatggctac 600tcttaccgag ggaaaatgaa taggacagtc aaccagcatg cgtgccttta ctggaactcc 660cacctcctct tgcaggagaa ttacaacatg tttatggagg atgctgaaac ccatgggatt 720ggggaacaca atttctgcag aaacccagat gcggacgaaa agccctggtg ctttattaaa 780gttaccaatg acaaggtgaa atgggaatac tgtgatgtct cagcctgctc agcccaggac 840gttgcctacc cagaggaaag ccccactgag ccatcaacca agcttccggg gtttgactcc 900tgtggaaaga ctgagatagc agagaggaag atcaagagaa tctatggagg ctttaagagc 960acggcgggca agcacccatg gcaggcgtcc ctccagtcct cgctgcctct gaccatctcc 1020atgccccagg gccacttctg tggtggggcg ctgatccacc cctgctgggt gctcactgct 1080gcccactgca ccgacataaa aaccagacat ctaaaggtgg tgctagggga ccaggacctg 1140aagaaagaag aatttcatga gcagagcttt agggtggaga agatattcaa gtacagccac 1200tacaatgaaa gagatgagat tccccacaat gatattgcat tgctcaagtt aaagccagtg 1260gatggtcact gtgctctaga atccaaatac gtgaagactg tgtgcttgcc tgatgggtcc 1320tttccctctg ggagtgagtg ccacatctct ggctggggtg ttacagaaac aggaaaaggg 1380tcccgccagc tcctggatgc caaagtcaag ctgattgcca acactttgtg caactcccgc 1440caactctatg accacatgat tgatgacagt atgatctgtg caggaaatct tcagaaacct 1500gggcaagaca cctgccaggg tgactctgga ggccccctga cctgtgagaa ggacggcacc 1560tactacgtct atgggatagt gagctggggc ctggagtgtg agaagaggcc aggggtctac 1620acccaagtta ccaaattcct gaattggatc aaagccacca tcaaaagtga aagtggcttc 1680taa 1683 3 1683 DNA Homo sapiens 3 atgtttgcca ggatgtctga tctccatgttctgctgttaa tggctctggt gggaaagaca 60 gcctgtgggt tctccctgat gtctttattggaaagcctgg acccagactg gacccctgac 120 cagtatgatt acagctacga ggattataatcaggaagaga acaccagtag cacacttacc 180 catgctgaga atcctgactg gtactacactgaggaccaag ctgatccatg ccagcccaac 240 ccctgtgaac acggtgggga ctgcctcgtccatgggagca ccttcacatg cagctgcctg 300 gctcctttct ctgggaataa gtgtcagaaagtgcaaaata cgtgcaagga caacccatgt 360 ggccggggcc aatgtctcat tacccagagtcctccctact accgctgtgt ctgtaaacac 420 ccttacacag gtcccagctg ctcccaagtggttcctgtat gcaggccaaa cccctgccag 480 aatggggcta cctgctcccg gcataagcggagatccaagt tcacctgtgc ctgtcccgac 540 cagttcaagg ggaaattctg tgaaataggttctgatgact gctatgttgg cgatggctac 600 tcttaccgag ggaaaatgaa taggacagtcaaccagcatg cgtgccttta ctggaactcc 660 cacctcctct tgcaggagaa ttacaacatgtttatggagg atgctgaaac ccatgggatt 720 ggggaacaca atttctgcag aaacccagatgcggacgaaa agccctggtg ctttattaaa 780 gttaccaatg acaaggtgaa atgggaatactgtgatgtct cagcctgctc agcccaggac 840 gttgcctacc cagaggaaag ccccactgagccatcaacca agcttccggg gtttgactcc 900 tgtggaaaga ctgagatagc agagaggaagatcaagagaa tctatggagg ctttaagagc 960 acggcgggca agcacccatg gcaggcgtccctccagtcct cgctgcctct gaccatctcc 1020 atgccccagg gccacttctg tggtggggcgctgatccacc cctgctgggt gctcactgct 1080 gcccactgca ccgacataaa aaccagacatctaaaggtgg tgctagggga ccaggacctg 1140 aagaaagaag aatttcatga gcagagctttagggtgcaga agatattcaa gtacagccac 1200 tacaatgaaa gagatgagat tccccacaatgatattgcat tgctcaagtt aaagccagtg 1260 gatggtcact gtgctctaga atccaaatacgtgaagactg tgtgcttgcc tgatgggtcc 1320 tttccctctg ggagtgagtg ccacatctctggctggggtg ttacagaaac aggaaaaggg 1380 tcccgccagc tcctggatgc caaagtcaagctgattgcca acactttgtg caactcccgc 1440 caactctatg accacatgat tgatgacagtatgatctgtg caggaaatct tcagaaacct 1500 gggcaagaca cctgccaggg tgactctggaggccccctga cctgtgagaa ggacggcacc 1560 tactacgtct atgggatagt gagctggggcctggagtgtg ggaagaggcc aggggtctac 1620 acccaagtta ccaaattcct gaattggatcaaagccacca tcaaaagtga aagtggcttc 1680 taa 1683 4 1683 DNA Homo sapiens 4atgtttgcca ggatgtctga tctccatgtt ctgctgttaa tggctctggt gggaaagaca 60gcctgtgggt tctccctgat gtctttattg gaaagcctgg acccagactg gacccctgac 120cagtatgatt acagctacga ggattataat caggaagaga acaccagtag cacacttacc 180catgctgaga atcctgactg gtactacact gaggaccaag ctgatccatg ccagcccaac 240ccctgtgaac acggtgggga ctgcctcgtc catgggagca ccttcacatg cagctgcctg 300gctcctttct ctgggaataa gtgtcagaaa gtgcaaaata cgtgcaagga caacccatgt 360ggccggggcc aatgtctcat tacccagagt cctccctact accgctgtgt ctgtaaacac 420ccttacacag gtcccagctg ctcccaagtg gttcctgtat gcaggccaaa cccctgccag 480aatggggcta cctgctcccg gcataagcgg agatccaagt tcacctgtgc ctgtcccgac 540cagttcaagg ggaaattctg tgaaataggt tctgatgact gctatgttgg cgatggctac 600tcttaccgag ggaaaatgaa taggacagtc aaccagcatg cgtgccttta ctggaactcc 660cacctcctct tgcaggagaa ttacaacatg tttatggagg atgctgaaac ccatgggatt 720ggggaacaca atttctgcag aaacccagat gcggacgaaa agccctggtg ctttattaaa 780gttaccaatg acaaggtgaa atgggaatac tgtgatgtct cagcctgctc agcccaggac 840gttgcctacc cagaggaaag ccccactgag ccatcaacca agcttccggg gtttgactcc 900tgtggaaaga ctgagatagc agagaggaag atcaagagaa tctatggagg ctttaagagc 960acggcgggca agcacccatg gcaggcgtcc ctccagtcct cgctgcctct gaccatctcc 1020atgccccagg gccacttctg tggtggggcg ctgatccacc cctgctgggt gctcactgct 1080gcccactgca ccgacataaa aaccagacat ctaaaggtgg tgctagggga ccaggacctg 1140aagaaagaag aatttcatga gcagagcttt agggtgcaga agatattcaa gtacagccac 1200tacaatgaaa gagatgagat tccccacaat gatattgcat tgctcaagtt aaagccagtg 1260gatggtcact gtgctctaga atccaaatac gtgaagactg tgtgcttgcc tgatgggtcc 1320tttccctctg ggagtgagtg ccacatctct ggctggggtg ttacagaaac aggaaaaggg 1380tcccgccagc tcctggatgc caaagtcaag ctgattgcca acactttgtg caactcccgc 1440caactctatg accacatgat tgatgacagt atgatctgtg caggaaatct tcagaaacct 1500gggcaagaca cctgccaggg tgactctgga ggccccctga cctgtgagaa ggacggcacc 1560tactacgtct atgggatagt gagctggggc ctggagtgtg agaagaggcc aggggtctac 1620acccaagtta ccaaattcct gaattggatc aaagccacca tcaaaagtga aagtggcttc 1680taa 1683 5 560 PRT Homo sapiens 5 Met Phe Ala Arg Met Ser Asp Leu HisVal Leu Leu Leu Met Ala Leu 1 5 10 15 Val Gly Lys Thr Ala Cys Gly PheSer Leu Met Ser Leu Leu Glu Ser 20 25 30 Leu Asp Pro Asp Trp Thr Pro AspGln Tyr Asp Tyr Ser Tyr Glu Asp 35 40 45 Tyr Asn Gln Glu Glu Asn Thr SerSer Thr Leu Thr His Ala Glu Asn 50 55 60 Pro Asp Trp Tyr Tyr Thr Glu AspGln Ala Asp Pro Cys Gln Pro Asn 65 70 75 80 Pro Cys Glu His Gly Gly AspCys Leu Val His Gly Ser Thr Phe Thr 85 90 95 Cys Ser Cys Leu Ala Pro PheSer Gly Asn Lys Cys Gln Lys Val Gln 100 105 110 Asn Thr Cys Lys Asp AsnPro Cys Gly Arg Gly Gln Cys Leu Ile Thr 115 120 125 Gln Ser Pro Pro TyrTyr Arg Cys Val Cys Lys His Pro Tyr Thr Gly 130 135 140 Pro Ser Cys SerGln Val Val Pro Val Cys Arg Pro Asn Pro Cys Gln 145 150 155 160 Asn GlyAla Thr Cys Ser Arg His Lys Arg Arg Ser Lys Phe Thr Cys 165 170 175 AlaCys Pro Asp Gln Phe Lys Gly Lys Phe Cys Glu Ile Gly Ser Asp 180 185 190Asp Cys Tyr Val Gly Asp Gly Tyr Ser Tyr Arg Gly Lys Met Asn Arg 195 200205 Thr Val Asn Gln His Ala Cys Leu Tyr Trp Asn Ser His Leu Leu Leu 210215 220 Gln Glu Asn Tyr Asn Met Phe Met Glu Asp Ala Glu Thr His Gly Ile225 230 235 240 Gly Glu His Asn Phe Cys Arg Asn Pro Asp Ala Asp Glu LysPro Trp 245 250 255 Cys Phe Ile Lys Val Thr Asn Asp Lys Val Lys Trp GluTyr Cys Asp 260 265 270 Val Ser Ala Cys Ser Ala Gln Asp Val Ala Tyr ProGlu Glu Ser Pro 275 280 285 Thr Glu Pro Ser Thr Lys Leu Pro Gly Phe AspSer Cys Gly Lys Thr 290 295 300 Glu Ile Ala Glu Arg Lys Ile Lys Arg IleTyr Gly Gly Phe Lys Ser 305 310 315 320 Thr Ala Gly Lys His Pro Trp GlnAla Ser Leu Gln Ser Ser Leu Pro 325 330 335 Leu Thr Ile Ser Met Pro GlnGly His Phe Cys Gly Gly Ala Leu Ile 340 345 350 His Pro Cys Trp Val LeuThr Ala Ala His Cys Thr Asp Ile Lys Thr 355 360 365 Arg His Leu Lys ValVal Leu Gly Asp Gln Asp Leu Lys Lys Glu Glu 370 375 380 Phe His Glu GlnSer Phe Arg Val Glu Lys Ile Phe Lys Tyr Ser His 385 390 395 400 Tyr AsnGlu Arg Asp Glu Ile Pro His Asn Asp Ile Ala Leu Leu Lys 405 410 415 LeuLys Pro Val Asp Gly His Cys Ala Leu Glu Ser Lys Tyr Val Lys 420 425 430Thr Val Cys Leu Pro Asp Gly Ser Phe Pro Ser Gly Ser Glu Cys His 435 440445 Ile Ser Gly Trp Gly Val Thr Glu Thr Gly Lys Gly Ser Arg Gln Leu 450455 460 Leu Asp Ala Lys Val Lys Leu Ile Ala Asn Thr Leu Cys Asn Ser Arg465 470 475 480 Gln Leu Tyr Asp His Met Ile Asp Asp Ser Met Ile Cys AlaGly Asn 485 490 495 Leu Gln Lys Pro Gly Gln Asp Thr Cys Gln Gly Asp SerGly Gly Pro 500 505 510 Leu Thr Cys Glu Lys Asp Gly Thr Tyr Tyr Val TyrGly Ile Val Ser 515 520 525 Trp Gly Leu Glu Cys Gly Lys Arg Pro Gly ValTyr Thr Gln Val Thr 530 535 540 Lys Phe Leu Asn Trp Ile Lys Ala Thr IleLys Ser Glu Ser Gly Phe 545 550 555 560 6 560 PRT Homo sapiens 6 Met PheAla Arg Met Ser Asp Leu His Val Leu Leu Leu Met Ala Leu 1 5 10 15 ValGly Lys Thr Ala Cys Gly Phe Ser Leu Met Ser Leu Leu Glu Ser 20 25 30 LeuAsp Pro Asp Trp Thr Pro Asp Gln Tyr Asp Tyr Ser Tyr Glu Asp 35 40 45 TyrAsn Gln Glu Glu Asn Thr Ser Ser Thr Leu Thr His Ala Glu Asn 50 55 60 ProAsp Trp Tyr Tyr Thr Glu Asp Gln Ala Asp Pro Cys Gln Pro Asn 65 70 75 80Pro Cys Glu His Gly Gly Asp Cys Leu Val His Gly Ser Thr Phe Thr 85 90 95Cys Ser Cys Leu Ala Pro Phe Ser Gly Asn Lys Cys Gln Lys Val Gln 100 105110 Asn Thr Cys Lys Asp Asn Pro Cys Gly Arg Gly Gln Cys Leu Ile Thr 115120 125 Gln Ser Pro Pro Tyr Tyr Arg Cys Val Cys Lys His Pro Tyr Thr Gly130 135 140 Pro Ser Cys Ser Gln Val Val Pro Val Cys Arg Pro Asn Pro CysGln 145 150 155 160 Asn Gly Ala Thr Cys Ser Arg His Lys Arg Arg Ser LysPhe Thr Cys 165 170 175 Ala Cys Pro Asp Gln Phe Lys Gly Lys Phe Cys GluIle Gly Ser Asp 180 185 190 Asp Cys Tyr Val Gly Asp Gly Tyr Ser Tyr ArgGly Lys Met Asn Arg 195 200 205 Thr Val Asn Gln His Ala Cys Leu Tyr TrpAsn Ser His Leu Leu Leu 210 215 220 Gln Glu Asn Tyr Asn Met Phe Met GluAsp Ala Glu Thr His Gly Ile 225 230 235 240 Gly Glu His Asn Phe Cys ArgAsn Pro Asp Ala Asp Glu Lys Pro Trp 245 250 255 Cys Phe Ile Lys Val ThrAsn Asp Lys Val Lys Trp Glu Tyr Cys Asp 260 265 270 Val Ser Ala Cys SerAla Gln Asp Val Ala Tyr Pro Glu Glu Ser Pro 275 280 285 Thr Glu Pro SerThr Lys Leu Pro Gly Phe Asp Ser Cys Gly Lys Thr 290 295 300 Glu Ile AlaGlu Arg Lys Ile Lys Arg Ile Tyr Gly Gly Phe Lys Ser 305 310 315 320 ThrAla Gly Lys His Pro Trp Gln Ala Ser Leu Gln Ser Ser Leu Pro 325 330 335Leu Thr Ile Ser Met Pro Gln Gly His Phe Cys Gly Gly Ala Leu Ile 340 345350 His Pro Cys Trp Val Leu Thr Ala Ala His Cys Thr Asp Ile Lys Thr 355360 365 Arg His Leu Lys Val Val Leu Gly Asp Gln Asp Leu Lys Lys Glu Glu370 375 380 Phe His Glu Gln Ser Phe Arg Val Glu Lys Ile Phe Lys Tyr SerHis 385 390 395 400 Tyr Asn Glu Arg Asp Glu Ile Pro His Asn Asp Ile AlaLeu Leu Lys 405 410 415 Leu Lys Pro Val Asp Gly His Cys Ala Leu Glu SerLys Tyr Val Lys 420 425 430 Thr Val Cys Leu Pro Asp Gly Ser Phe Pro SerGly Ser Glu Cys His 435 440 445 Ile Ser Gly Trp Gly Val Thr Glu Thr GlyLys Gly Ser Arg Gln Leu 450 455 460 Leu Asp Ala Lys Val Lys Leu Ile AlaAsn Thr Leu Cys Asn Ser Arg 465 470 475 480 Gln Leu Tyr Asp His Met IleAsp Asp Ser Met Ile Cys Ala Gly Asn 485 490 495 Leu Gln Lys Pro Gly GlnAsp Thr Cys Gln Gly Asp Ser Gly Gly Pro 500 505 510 Leu Thr Cys Glu LysAsp Gly Thr Tyr Tyr Val Tyr Gly Ile Val Ser 515 520 525 Trp Gly Leu GluCys Glu Lys Arg Pro Gly Val Tyr Thr Gln Val Thr 530 535 540 Lys Phe LeuAsn Trp Ile Lys Ala Thr Ile Lys Ser Glu Ser Gly Phe 545 550 555 560 7560 PRT Homo sapiens 7 Met Phe Ala Arg Met Ser Asp Leu His Val Leu LeuLeu Met Ala Leu 1 5 10 15 Val Gly Lys Thr Ala Cys Gly Phe Ser Leu MetSer Leu Leu Glu Ser 20 25 30 Leu Asp Pro Asp Trp Thr Pro Asp Gln Tyr AspTyr Ser Tyr Glu Asp 35 40 45 Tyr Asn Gln Glu Glu Asn Thr Ser Ser Thr LeuThr His Ala Glu Asn 50 55 60 Pro Asp Trp Tyr Tyr Thr Glu Asp Gln Ala AspPro Cys Gln Pro Asn 65 70 75 80 Pro Cys Glu His Gly Gly Asp Cys Leu ValHis Gly Ser Thr Phe Thr 85 90 95 Cys Ser Cys Leu Ala Pro Phe Ser Gly AsnLys Cys Gln Lys Val Gln 100 105 110 Asn Thr Cys Lys Asp Asn Pro Cys GlyArg Gly Gln Cys Leu Ile Thr 115 120 125 Gln Ser Pro Pro Tyr Tyr Arg CysVal Cys Lys His Pro Tyr Thr Gly 130 135 140 Pro Ser Cys Ser Gln Val ValPro Val Cys Arg Pro Asn Pro Cys Gln 145 150 155 160 Asn Gly Ala Thr CysSer Arg His Lys Arg Arg Ser Lys Phe Thr Cys 165 170 175 Ala Cys Pro AspGln Phe Lys Gly Lys Phe Cys Glu Ile Gly Ser Asp 180 185 190 Asp Cys TyrVal Gly Asp Gly Tyr Ser Tyr Arg Gly Lys Met Asn Arg 195 200 205 Thr ValAsn Gln His Ala Cys Leu Tyr Trp Asn Ser His Leu Leu Leu 210 215 220 GlnGlu Asn Tyr Asn Met Phe Met Glu Asp Ala Glu Thr His Gly Ile 225 230 235240 Gly Glu His Asn Phe Cys Arg Asn Pro Asp Ala Asp Glu Lys Pro Trp 245250 255 Cys Phe Ile Lys Val Thr Asn Asp Lys Val Lys Trp Glu Tyr Cys Asp260 265 270 Val Ser Ala Cys Ser Ala Gln Asp Val Ala Tyr Pro Glu Glu SerPro 275 280 285 Thr Glu Pro Ser Thr Lys Leu Pro Gly Phe Asp Ser Cys GlyLys Thr 290 295 300 Glu Ile Ala Glu Arg Lys Ile Lys Arg Ile Tyr Gly GlyPhe Lys Ser 305 310 315 320 Thr Ala Gly Lys His Pro Trp Gln Ala Ser LeuGln Ser Ser Leu Pro 325 330 335 Leu Thr Ile Ser Met Pro Gln Gly His PheCys Gly Gly Ala Leu Ile 340 345 350 His Pro Cys Trp Val Leu Thr Ala AlaHis Cys Thr Asp Ile Lys Thr 355 360 365 Arg His Leu Lys Val Val Leu GlyAsp Gln Asp Leu Lys Lys Glu Glu 370 375 380 Phe His Glu Gln Ser Phe ArgVal Gln Lys Ile Phe Lys Tyr Ser His 385 390 395 400 Tyr Asn Glu Arg AspGlu Ile Pro His Asn Asp Ile Ala Leu Leu Lys 405 410 415 Leu Lys Pro ValAsp Gly His Cys Ala Leu Glu Ser Lys Tyr Val Lys 420 425 430 Thr Val CysLeu Pro Asp Gly Ser Phe Pro Ser Gly Ser Glu Cys His 435 440 445 Ile SerGly Trp Gly Val Thr Glu Thr Gly Lys Gly Ser Arg Gln Leu 450 455 460 LeuAsp Ala Lys Val Lys Leu Ile Ala Asn Thr Leu Cys Asn Ser Arg 465 470 475480 Gln Leu Tyr Asp His Met Ile Asp Asp Ser Met Ile Cys Ala Gly Asn 485490 495 Leu Gln Lys Pro Gly Gln Asp Thr Cys Gln Gly Asp Ser Gly Gly Pro500 505 510 Leu Thr Cys Glu Lys Asp Gly Thr Tyr Tyr Val Tyr Gly Ile ValSer 515 520 525 Trp Gly Leu Glu Cys Gly Lys Arg Pro Gly Val Tyr Thr GlnVal Thr 530 535 540 Lys Phe Leu Asn Trp Ile Lys Ala Thr Ile Lys Ser GluSer Gly Phe 545 550 555 560 8 560 PRT Homo sapiens 8 Met Phe Ala Arg MetSer Asp Leu His Val Leu Leu Leu Met Ala Leu 1 5 10 15 Val Gly Lys ThrAla Cys Gly Phe Ser Leu Met Ser Leu Leu Glu Ser 20 25 30 Leu Asp Pro AspTrp Thr Pro Asp Gln Tyr Asp Tyr Ser Tyr Glu Asp 35 40 45 Tyr Asn Gln GluGlu Asn Thr Ser Ser Thr Leu Thr His Ala Glu Asn 50 55 60 Pro Asp Trp TyrTyr Thr Glu Asp Gln Ala Asp Pro Cys Gln Pro Asn 65 70 75 80 Pro Cys GluHis Gly Gly Asp Cys Leu Val His Gly Ser Thr Phe Thr 85 90 95 Cys Ser CysLeu Ala Pro Phe Ser Gly Asn Lys Cys Gln Lys Val Gln 100 105 110 Asn ThrCys Lys Asp Asn Pro Cys Gly Arg Gly Gln Cys Leu Ile Thr 115 120 125 GlnSer Pro Pro Tyr Tyr Arg Cys Val Cys Lys His Pro Tyr Thr Gly 130 135 140Pro Ser Cys Ser Gln Val Val Pro Val Cys Arg Pro Asn Pro Cys Gln 145 150155 160 Asn Gly Ala Thr Cys Ser Arg His Lys Arg Arg Ser Lys Phe Thr Cys165 170 175 Ala Cys Pro Asp Gln Phe Lys Gly Lys Phe Cys Glu Ile Gly SerAsp 180 185 190 Asp Cys Tyr Val Gly Asp Gly Tyr Ser Tyr Arg Gly Lys MetAsn Arg 195 200 205 Thr Val Asn Gln His Ala Cys Leu Tyr Trp Asn Ser HisLeu Leu Leu 210 215 220 Gln Glu Asn Tyr Asn Met Phe Met Glu Asp Ala GluThr His Gly Ile 225 230 235 240 Gly Glu His Asn Phe Cys Arg Asn Pro AspAla Asp Glu Lys Pro Trp 245 250 255 Cys Phe Ile Lys Val Thr Asn Asp LysVal Lys Trp Glu Tyr Cys Asp 260 265 270 Val Ser Ala Cys Ser Ala Gln AspVal Ala Tyr Pro Glu Glu Ser Pro 275 280 285 Thr Glu Pro Ser Thr Lys LeuPro Gly Phe Asp Ser Cys Gly Lys Thr 290 295 300 Glu Ile Ala Glu Arg LysIle Lys Arg Ile Tyr Gly Gly Phe Lys Ser 305 310 315 320 Thr Ala Gly LysHis Pro Trp Gln Ala Ser Leu Gln Ser Ser Leu Pro 325 330 335 Leu Thr IleSer Met Pro Gln Gly His Phe Cys Gly Gly Ala Leu Ile 340 345 350 His ProCys Trp Val Leu Thr Ala Ala His Cys Thr Asp Ile Lys Thr 355 360 365 ArgHis Leu Lys Val Val Leu Gly Asp Gln Asp Leu Lys Lys Glu Glu 370 375 380Phe His Glu Gln Ser Phe Arg Val Gln Lys Ile Phe Lys Tyr Ser His 385 390395 400 Tyr Asn Glu Arg Asp Glu Ile Pro His Asn Asp Ile Ala Leu Leu Lys405 410 415 Leu Lys Pro Val Asp Gly His Cys Ala Leu Glu Ser Lys Tyr ValLys 420 425 430 Thr Val Cys Leu Pro Asp Gly Ser Phe Pro Ser Gly Ser GluCys His 435 440 445 Ile Ser Gly Trp Gly Val Thr Glu Thr Gly Lys Gly SerArg Gln Leu 450 455 460 Leu Asp Ala Lys Val Lys Leu Ile Ala Asn Thr LeuCys Asn Ser Arg 465 470 475 480 Gln Leu Tyr Asp His Met Ile Asp Asp SerMet Ile Cys Ala Gly Asn 485 490 495 Leu Gln Lys Pro Gly Gln Asp Thr CysGln Gly Asp Ser Gly Gly Pro 500 505 510 Leu Thr Cys Glu Lys Asp Gly ThrTyr Tyr Val Tyr Gly Ile Val Ser 515 520 525 Trp Gly Leu Glu Cys Glu LysArg Pro Gly Val Tyr Thr Gln Val Thr 530 535 540 Lys Phe Leu Asn Trp IleLys Ala Thr Ile Lys Ser Glu Ser Gly Phe 545 550 555 560

What is claimed is:
 1. An atherothrombosis risk factor which comprises acoagulation factor VII-activating protease (FSAP) mutant.
 2. The riskfactor as claimed in claim 1, wherein the FSAP mutant comprises a Gly toGlu exchange at amino acid position 534 of the proenzyme sequence. 3.The risk factor as claimed in claim 2, wherein the FSAP mutant furthercomprises a Glu to Gln exchange at amino acid position 393 of theproenzyme sequence.
 4. The risk factor as claimed in claim 1, whereinthe FSAP mutant is encoded by a proenzyme nucleotide sequence comprisinga G to A base exchange at position
 1601. 5. The risk factor as claimedin claim 4, wherein the FSAP mutant is encoded by a proenzyme nucleotidesequence further comprising a G to C base exchange at position
 1177. 6.The risk factor as claimed in claim 1, wherein the FSAP mutant haspartially or completely lost the ability to activate single-chainplasminogen activators, as compared with wild-type FSAP.
 7. The riskfactor as claimed in claim 1, wherein the FSAP mutant has partially orcompletely lost the ability to activate prourokinase, as compared withwild-type FSAP.
 8. The risk factor as claimed in claim 1, whichindicates a genetic predisposition to the development of atheroscleroticdisorders and their sequelae, or thrombotic disorders and theirsequelae.
 9. The risk factor as claimed in claim 8, which indicates agenetic predisposition to the development of at least one of arterialand venous occlusive disorders.
 10. The risk factor as claimed in claim8, which indicates a genetic predisposition to the development of atleast one of atherosclerotic and thrombotic restrictions of organfunctions.
 11. The risk factor as claimed in claim 8, which indicates agenetic predisposition to the development of one or more of anginapectoris, myocardial infarction, and strokes.
 12. An atherothrombosisrisk factor, wherein the potential for activation of single-chainplasminogen activators is reduced.
 13. The atherothrombosis risk factoras claimed in claim 12, wherein the potential for activation ofsingle-chain plasminogen activators is reduced by a diminished FSAPactivity in at least one of whole blood and blood plasma.
 14. Theatherothrombosis risk factor as claimed in claim 12, wherein thepotential for activation of prourokinase is reduced.
 15. A diagnosticmethod for identifying a genetic predisposition to the development of atleast one of atherosclerosis and thromboses, which comprises determiningat least one of a reduced FSAP protein concentration and a reduced FSAPprourokinase activating activity in one or more body fluids of anindividual.
 16. A diagnostic method as claimed in claim 15, wherein theratio between FSAP protein concentration and FSAP prourokinaseactivating potency in one or more body fluids of an individual isdetermined to identify the genetic predisposition.
 17. The diagnosticmethod as claimed in claim 15, wherein the one or more body fluidscomprise blood plasma.
 18. The diagnostic method as claimed in claim 15,wherein the potential for activation of single-chain plasminogenactivators is determined in one or more body fluids of the individual.19. The diagnostic method as claimed in claim 18, wherein the potentialfor activation of prourokinase is determined in one or more body fluidsof the individual.
 20. The diagnostic method as claimed in claim 15,further comprising detection of heterozygous and/or homozygous mutantsof the FSAP proenzyme nucleotide sequence with a G to A base exchange atnucleotide position 1601 by analysis of at least one of genomic DNA,mRNA, and cDNA of the individual.
 21. The diagnostic method as claimedin claim 15, further comprising detection of heterozygous and/orhomozygous mutants of the FSAP proenzyme nucleotide sequence with a G toC base exchange at nucleotide position
 1177. 22. The diagnostic methodas claimed in claim 15, comprising detecting FSAP mutants at the proteinlevel.
 23. The diagnostic method as claimed in claim 22, wherein theFSAP mutants are detected by using FSAP-specific and/or mutantFSAP-specific antibodies.
 24. The diagnostic method as claimed in claim15, wherein the FSAP mutants are detected by histological investigationson at least one of tissues and in solutions extracted from tissues. 25.The diagnostic method as claimed in claim 15, comprising one or more of:(a) incubating a sample that could contain one or more FSAP mutants witha first antibody, immobilized on a solid support, then, after washing,adding a second, labeled antibody and, after washing out again,measuring the signal produced by the second antibody, wherein the secondantibody may comprise a wild-type FSAP-specific antibody; (b) incubatinga sample that could contain one or more FSAP mutants with a firstantibody immobilized on a solid support, then, after washing, adding asecond, labeled antibody and, after washing out again, measuring thesignal produced by the second antibody, wherein the first antibody is awild-type FSAP-specific antibody; (c) immobilizing a sample that couldcontain one or more FSAP mutants on a support and detecting the samplewith a labeled antibody, alone or in a mixture with an unlabelledantibody; and (d) incubating a sample that could contain one or moreFSAP mutants with an antibody immobilized on a support in the presenceof a labeled FSAP mutant, and measuring the signal produced by thelabel.
 26. The diagnostic method as claimed in claim 15, wherein FSAPactivity is measured by: (a) incubating a sample that could contain oneor more FSAP mutants on a solid support onto which an antibody haspreviously been coupled, wherein the antibody is at least one of anFSAP-specific, a wild-type FSAP-specific, and a mutant FSAP-specificantibody; and (b) after washing out the free support, incubating theFSAP immobilized thereon with reagents which allow determination of theactivity thereof.
 27. The diagnostic method as claimed in claim 15,wherein antibodies are used for detection of FSAP mutants by one or moreof Western blots, immunohistology, and fluorescence-activated cellsorting (FACS).
 28. A test system for carrying out the diagnostic methodas claimed in claim
 15. 29. A diagnostic method for detecting antibodiesagainst at least one of factor VII-activating protease (FSAP) and FSAPmutants, which comprises allowing a sample that could contain theantibodies to act on at least one of FSAP and FSAP mutants immobilizedon a solid support, then washing and detecting the antibodies bound tothe solid support.
 30. The diagnostic method as claimed in claim 29,wherein the antibodies bound to the solid support are incubated with asubstance selected from one or more of labelled anti-humanimmunoglobulin, fragments thereof, labelled protein A, and labelledprotein G; wherein the signal emitted by the bound, labelled substanceis determined.
 31. The diagnostic method as claimed in claim 29, whereinthe antibodies bound to the solid support are detected by a photometricmeasurement of the extinction caused by cleavage of a suitablechromogenic or fluorogenic substrate by one or more of enzyme-coupledanti-human antibodies, fragments thereof, protein A, and protein G. 32.The diagnostic method as claimed in claim 29, wherein the antibodiesbound to the solid support are detected by fluorescence measurement. 33.The diagnostic method as claimed in claim 29, wherein the antibodiesbound to the solid support are detected by radiometric measurement.